The most common form of congenital adrenal hyperplasia, 21-hydroxylase deficiency (21OHD), is also one of the most common genetic diseases. A hallmark characteristic of all patients with 21OHD is adrenal androgen excess. These androgens derive from accumulation of precursors, such as 17-hydroxyprogesterone (17OHP4), proximal to the defective enzymatic step, which are converted to testosterone (T) via accessible pathways involving steroid 17-hydroxylase/17, 20-lyase (CYP17A1) under the stimulation of cofactor protein cytochrome b5 (CYB5A). Dehydroepiandrosterone (DHEA), its sulfate (DHEAS) and androstenedione (AD) are thought to be the major androgen precursors of adrenal origin. Although serum 17OHP4 has long been used to monitor treatment of CAH, it correlates poorly with DHEA and AD; furthermore, no good correlation between these routinely measured androgens and clinical evidence of androgen excess has been demonstrated. In addition, routine measurement of AD and T in adults with 21OHD can be confusing, because these steroids also derive from the gonads. A biomarker of androgen production unique to the adrenal gland would be a major advance in the management of 21OHD. We propose as biomarker candidate's 11b-hydroxylated 19- carbon steroids and their 11-keto metabolites, because 11b-hydroxylase (CYP11B1) is expressed only in the adrenal gland and not in the gonad. In addition, other androgens and precursors might be elevated in patients with 21OHD. Recent data has shown elevation of metabolites from the so-called backdoor pathway to dihydrotestosterone (DHT) in patients with 21OHD. These intermediates emerge via an enzymatic cascade initiated by the 5?-reduction of 17OHP4, which bypasses the intermediacy of DHEA, AD, or T to the most potent androgen, DHT. In addition, we hypothesize that the abundance of D4-androgens in these patients is facilitated by disrupted adrenal expression of androgenic enzymes and cofactors, conferring the adrenal a gonadal-like zone. The proposed studies aim to (1) determine the zonal distribution of key androgenic enzymes and cofactors within the adrenal cortex in patients with 21OHD and (2) to characterize in detail the adrenal androgen and intermediates in 21OHD, which derive via traditional and novel pathways, with the ultimate goal of identifying adrenal-specific biomarkers of androgen excess. In Aim 1, we will immunostain normal adrenal tissue and adrenal glands obtained from patients with 21OHD and ACTH-dependent Cushing syndrome for CYB5A and 3b-hydroxysteroid dehydrogenase type 2 (HSD3B2), two enzymes necessary for androgen synthesis, but normally expressed in different adrenal zones. We hypothesize that 21OHD and chronic ACTH stimulation disrupt the normal adrenal enzyme distribution, contributing to excessive androgen synthesis. In Aim 2, we will characterize steroid profiles using liquid chromatography-tandem mass spectrometry analysis of blood samples obtained from patients with classic and non-classic 21OHD and from normal controls.